Recombinant Escherichia fergusonii Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the UbiB protein and what is its role in ubiquinone biosynthesis?

UbiB is a conserved protein involved in the biosynthesis of ubiquinone (UQ), also known as coenzyme Q, which plays a crucial role in cellular bioenergetics. UbiB possesses ATPase activity and functions as an essential component in the ubiquinone biosynthetic pathway . The protein belongs to the UbiB family, characterized by a protein kinase-like (PKL) domain, though the exact mechanistic function of this domain remains under investigation . In E. coli and likely in E. fergusonii, UbiB contributes to the production of ubiquinone, which serves as the terminal electron acceptor in aerobic respiration and is critical for cellular energy metabolism .

How does UbiB from E. fergusonii compare structurally to UbiB from E. coli?

E. fergusonii UbiB shares significant structural similarities with its E. coli homolog, as both are members of the highly conserved UbiB family. Both proteins contain the characteristic protein kinase-like domain that defines this family . Phylogenetic analysis suggests E. fergusonii is closely related to E. coli, which indicates conservation of essential metabolic pathways, including ubiquinone biosynthesis. The protein kinase-like domain in UbiB family proteins contains conserved amino acid residues (similar to K275, D288, and E330 identified in Cqd1) that are critical for ATP binding and function . While specific crystallographic data comparing both proteins is limited, sequence alignment analyses would likely reveal high homology in functional domains between the two species.

What are the oxygen-dependent and oxygen-independent pathways for ubiquinone biosynthesis in Escherichia species?

Escherichia species have evolved two distinct pathways for ubiquinone biosynthesis to adapt to environments with varying oxygen levels:

• Oxygen-dependent pathway: The traditional pathway requires molecular oxygen (O₂) as a substrate. It involves a series of reactions to modify the aromatic ring of 4-hydroxybenzoic acid (4-HB) through one prenylation, one decarboxylation, three hydroxylation, and three methylation reactions . The oxygen-dependent hydroxylation steps are crucial in this pathway.

• Oxygen-independent pathway: This novel pathway functions without requiring oxygen and relies on three essential proteins: UbiT (YhbT), UbiU (YhbU), and UbiV (YhbV). UbiT contains an SCP2 lipid-binding domain and likely serves as an accessory factor, while UbiU and UbiV form a heterodimer that functions as an O₂-independent hydroxylase. Each of these proteins binds a 4Fe-4S cluster via conserved cysteines that are essential for their activity .

This dual pathway system allows proteobacteria like Escherichia species to synthesize ubiquinone across the entire range of environmental oxygen conditions, demonstrating remarkable metabolic plasticity .

How do the iron-sulfur clusters in UbiU-UbiV complex function in the oxygen-independent hydroxylation during ubiquinone biosynthesis?

The UbiU-UbiV complex represents a novel class of oxygen-independent hydroxylases that are essential for ubiquinone biosynthesis under anaerobic conditions. Each protein in this heterodimer binds a 4Fe-4S cluster through conserved cysteine residues . These iron-sulfur clusters are critical for the catalytic activity of the complex.

In the absence of molecular oxygen, the 4Fe-4S clusters likely serve as electron transfer centers that facilitate the activation of alternative oxidants for hydroxylation reactions. The mechanism may involve:

• Initial binding of the substrate to the UbiU-UbiV complex

• Electron transfer from the substrate to the 4Fe-4S clusters

• Generation of a substrate radical intermediate

• Hydroxylation through an alternative oxygen donor (possibly water or a metabolic intermediate)

• Release of the hydroxylated product

This mechanism represents a significant departure from conventional oxygen-dependent hydroxylases and demonstrates the evolutionary adaptation of proteobacteria to fluctuating oxygen environments. Mutations in the conserved cysteine residues that coordinate the iron-sulfur clusters abolish the hydroxylase activity, confirming their essential role in the catalytic mechanism .

What functional domains and motifs are present in the recombinant E. fergusonii UbiB protein, and how do they contribute to its enzymatic activity?

The recombinant E. fergusonii UbiB protein contains several key functional domains and motifs:

• Protein Kinase-Like (PKL) Domain: The central feature of UbiB family proteins, this domain adopts a structure similar to protein kinases but has distinct functions in ubiquinone biosynthesis . The PKL domain likely binds ATP, though it may not catalyze typical phosphorylation reactions.

• Conserved Catalytic Residues: Similar to other UbiB family members, E. fergusonii UbiB contains conserved lysine (K), aspartate (D), and glutamate (E) residues that are critical for ATP binding and hydrolysis . These residues form part of the catalytic core.

• Membrane-Associated Regions: UbiB proteins typically contain hydrophobic regions that facilitate association with the inner mitochondrial or bacterial membrane, positioning them appropriately for interaction with the lipid-soluble intermediates of ubiquinone biosynthesis.

• Interaction Interfaces: Surfaces that mediate interactions with other ubiquinone biosynthesis proteins, particularly those involved in forming the multiprotein biosynthetic complex.

These domains collectively enable UbiB to function within the ubiquinone biosynthetic pathway, likely by coupling ATP hydrolysis to critical steps in the modification of ubiquinone precursors. Site-directed mutagenesis studies of conserved residues within the PKL domain, similar to those performed with Cqd1 (K275A, D288A, and E330A), would be expected to significantly reduce or abolish UbiB activity .

What is the relationship between UbiB and membrane phospholipid homeostasis in Escherichia species?

UbiB family proteins have been implicated in both ubiquinone biosynthesis and membrane phospholipid homeostasis, suggesting a multifunctional role for these proteins. Evidence for this relationship includes:

• Genetic Interactions: Members of the UbiB family show genetic interactions with proteins involved in phospholipid metabolism. For example, deletion of both Cqd1 (a UbiB family member) and Ups1 (responsible for phosphatidic acid transport) results in severe growth defects, indicating a functional relationship between UbiB family proteins and phospholipid pathways .

• Membrane Contact Sites: UbiB family members like Cqd1 can form novel membrane contact sites, potentially facilitating phospholipid transfer between membranes . This suggests a structural role in organizing membrane architecture.

• Mitochondrial Membrane Homeostasis: UbiB family members have been implicated in maintaining mitochondrial membrane integrity and morphology . The proper distribution of phospholipids is essential for these functions.

What are the optimal expression systems and purification protocols for producing recombinant E. fergusonii UbiB protein?

Expression Systems:

• E. coli BL21(DE3): The preferred expression system due to its high expression levels and genetic tractability. For optimal expression, consider the following parameters:

  • Induction with 0.5 mM IPTG at OD₆₀₀ of 0.6-0.8

  • Post-induction growth at 18°C for 16-18 hours to enhance protein folding

  • Supplementation with iron and sulfur sources if iron-sulfur cluster assembly is required

• Cell-Free Expression System: For proteins that may be toxic when overexpressed in bacteria, cell-free systems can be advantageous.

Purification Protocol:

• Cell Lysis: Sonication or high-pressure homogenization in buffer containing:

  • 50 mM Tris-HCl, pH 8.0

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

• Initial Purification: Affinity chromatography using:

  • Ni-NTA column for His-tagged protein

  • Elution with 250 mM imidazole gradient

• Secondary Purification:

  • Size exclusion chromatography using Superdex 200 column

  • Buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Protein Quality Assessment:

  • SDS-PAGE for purity analysis

  • Western blot for identity confirmation

  • Dynamic light scattering for homogeneity analysis

For functional studies, it's crucial to verify the integrity of the protein kinase-like domain and ensure any cofactors (such as ATP or metal ions) are properly incorporated during or after purification.

How can researchers effectively analyze the O₂-dependent and O₂-independent activities of UbiB in ubiquinone biosynthesis?

To effectively analyze UbiB activities in both oxygen-dependent and oxygen-independent ubiquinone biosynthesis pathways, researchers should employ a multi-faceted approach:

Anaerobic Techniques:

• Controlled Atmosphere Chambers: Use anaerobic glove boxes with controlled O₂ levels (0-0.1 ppm) for handling cultures and performing enzyme assays under strictly anaerobic conditions.

• Oxygen Scavenging Systems: Incorporate enzymatic oxygen scavenging systems (glucose oxidase/catalase or protocatechuate dioxygenase/protocatechuate) in reaction buffers.

Activity Assays:

• HPLC-MS Detection: Employ sensitive HPLC-MS methods to quantify ubiquinone and biosynthetic intermediates. This approach can detect even trace amounts of ubiquinone produced under various conditions .

• Isotope Labeling: Use ¹³C or ¹⁸O labeled precursors to track incorporation into ubiquinone molecules and determine the source of oxygen atoms in hydroxylation reactions.

• ATP Hydrolysis Assays: Measure ATPase activity using colorimetric phosphate release assays or coupled enzyme systems to assess UbiB function.

Genetic Approaches:

• Complementation Studies: Perform cross-complementation between E. coli and E. fergusonii UbiB in respective deletion mutants to assess functional conservation.

• Double Deletion Analysis: Create double deletions of UbiB with components of either O₂-dependent (e.g., UbiH) or O₂-independent (e.g., UbiU/UbiV) pathways to delineate pathway-specific roles .

Table 1: Comparative Analysis of O₂-Dependent and O₂-Independent Ubiquinone Biosynthesis Pathways

This comparative approach allows researchers to dissect the specific contributions of UbiB to each pathway and understand how these pathways coordinate in response to varying oxygen levels.

What techniques are most effective for studying the interactions between UbiB and other proteins in the ubiquinone biosynthesis complex?

Several complementary techniques can effectively characterize the interactions between UbiB and other proteins in the ubiquinone biosynthesis complex:

In Vitro Approaches:

• Co-Immunoprecipitation (Co-IP): Using specific antibodies against UbiB or epitope-tagged versions to pull down interaction partners from cell lysates, followed by mass spectrometry identification.

• Surface Plasmon Resonance (SPR): For quantitative assessment of binding affinities, association/dissociation rates, and interaction kinetics between purified UbiB and potential partners.

• Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters of protein-protein interactions, providing insights into binding energetics.

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): To analyze complex formation and determine the stoichiometry of multi-protein assemblies.

In Vivo Approaches:

• Förster Resonance Energy Transfer (FRET): Using fluorescently-tagged proteins to detect interactions in living cells.

• Bacterial Two-Hybrid Assay: Adapted for membrane proteins to screen for potential interaction partners.

• Chemical Cross-Linking coupled with Mass Spectrometry (XL-MS): To capture transient interactions and identify interaction interfaces.

• Proximity-Dependent Biotin Identification (BioID): For detecting both stable and transient protein interactions in their native cellular context.

Structural Approaches:

• Cryo-Electron Microscopy: For structural characterization of the entire biosynthetic complex.

• X-ray Crystallography: For high-resolution structures of UbiB in complex with specific partner proteins.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): To map interaction interfaces and conformational changes upon complex formation.

These techniques should be applied in combination to build a comprehensive understanding of how UbiB interacts with other proteins in both the O₂-dependent and O₂-independent ubiquinone biosynthesis pathways, potentially revealing differences in complex formation under varying oxygen conditions.

How should researchers interpret contradictory results when studying E. fergusonii UbiB in different experimental systems?

When facing contradictory results in E. fergusonii UbiB studies across different experimental systems, researchers should employ a structured approach to data interpretation:

Systematic Contradiction Analysis:

• Parameter Classification: Categorize contradictions using the (α, β, θ) notation system, where α represents the number of interdependent items, β represents the number of contradictory dependencies defined by domain experts, and θ represents the minimal number of required Boolean rules to assess these contradictions . This systematic approach helps to handle the complexity of multidimensional interdependencies within experimental datasets.

• Experimental System Comparison: Create a comparative matrix of all experimental variables:

  • Expression systems (E. coli vs. cell-free vs. native)

  • Growth conditions (aerobic vs. anaerobic)

  • Protein tags and their positions

  • Buffer compositions and pH values

  • Presence of detergents or membrane mimetics

• Functional Context Assessment: Evaluate whether contradictions arise from studying UbiB in isolation versus within its native multiprotein complex. The function of UbiB likely depends on interactions with other ubiquinone biosynthesis proteins and may differ between oxygen-dependent and oxygen-independent pathways.

• Species-Specific Differences: Although E. fergusonii is closely related to E. coli, subtle differences in regulatory elements or protein interfaces might lead to functional divergence. For example, studies on small RNAs in E. fergusonii have shown that despite high sequence conservation with E. coli, functional differences exist due to insertions or structural variations .

Resolution Strategies:

• Bridging Experiments: Design experiments that systematically bridge contradictory conditions to identify specific variables causing discrepancies.

• Multiple Orthogonal Techniques: Verify key findings using at least three independent methodological approaches.

• In Vivo Validation: Confirm biochemical findings with genetic complementation studies in UbiB deletion strains under both aerobic and anaerobic conditions.

• Controlled Variable Isolation: When testing a specific hypothesis, minimize system variations by standardizing all other parameters across experimental conditions.

By applying these structured approaches to contradiction analysis, researchers can transform apparent inconsistencies into valuable insights about context-dependent functions of E. fergusonii UbiB.

What are the key considerations when analyzing the kinetics of UbiB-catalyzed reactions in ubiquinone biosynthesis?

When analyzing the kinetics of UbiB-catalyzed reactions in ubiquinone biosynthesis, researchers should consider several critical factors:

Substrate Considerations:

• Lipophilic Nature: Ubiquinone precursors are highly hydrophobic molecules that partition into membranes. Kinetic analyses must account for substrate partitioning between aqueous and membrane phases.

• Substrate Accessibility: In native systems, substrates may be presented to UbiB through protein-protein interactions within a multiprotein complex. In reconstituted systems, the use of detergents or artificial membranes may alter substrate accessibility.

• Authentic Substrates: When possible, use authentic biosynthetic intermediates rather than synthetic analogs, as UbiB may exhibit different kinetics with non-native substrates.

Assay Design:

• ATP Hydrolysis Coupling: Since UbiB possesses ATPase activity , researchers should determine whether ATP hydrolysis is tightly coupled to substrate modification or if these activities can be uncoupled under certain conditions.

• Steady-State vs. Pre-Steady-State Kinetics: Pre-steady-state kinetic analyses using rapid mixing techniques may reveal transient intermediates or conformational changes that are not detectable in steady-state assays.

• Product Detection Methods: Develop sensitive analytical methods (HPLC-MS/MS) capable of detecting and quantifying both the disappearance of substrates and the appearance of products, as product inhibition may significantly affect kinetics.

Environmental Factors:

• Oxygen Sensitivity: The presence or absence of oxygen may alter UbiB activity or switch its function between different biosynthetic pathways . Carefully control oxygen levels during kinetic experiments.

• Redox State: The redox environment may affect UbiB activity, particularly if the protein contains or interacts with redox-sensitive cofactors. Include redox buffers of defined potential in reaction mixtures.

• Membrane Environment: The lipid composition of membranes can significantly influence membrane protein activity. Systematically vary lipid composition in reconstituted systems to determine optimal conditions.

Kinetic Model Selection:

• Complex Kinetic Models: Standard Michaelis-Menten kinetics may not adequately describe UbiB activity. Consider more complex models that account for:

  • Multiple substrates (ATP and ubiquinone precursors)

  • Membrane partitioning effects

  • Potential cooperativity or allostery

  • Product inhibition

• Global Data Fitting: When possible, fit multiple datasets simultaneously to discriminate between alternative kinetic models and constrain parameter values.

By addressing these considerations, researchers can develop more accurate kinetic models of UbiB function and better understand its role in ubiquinone biosynthesis.

How can researchers effectively investigate the role of UbiB in antibiotic resistance and virulence of E. fergusonii?

Investigating UbiB's role in antibiotic resistance and virulence of E. fergusonii requires a multifaceted approach that connects ubiquinone biosynthesis to bacterial survival strategies:

Genotype-Phenotype Correlation:

• UbiB Knockout and Complementation: Generate UbiB deletion mutants in E. fergusonii and complement with wild-type or site-directed mutants to assess:

  • Growth rates under various antibiotic stresses

  • Survival during oxidative stress challenges

  • Virulence in appropriate infection models

• Gene Expression Analysis: Quantify expression levels of UbiB under:

  • Antibiotic exposure conditions

  • Various oxygen tensions

  • Host-mimicking environments

  • In vivo infection settings

Antibiotic Resistance Mechanisms:

• Membrane Integrity Assessment: Since UbiB affects membrane homeostasis , measure:

  • Membrane potential using fluorescent indicators

  • Membrane permeability to hydrophobic and hydrophilic compounds

  • Lipid composition changes in UbiB mutants

• Resistance Profiling: Compare minimum inhibitory concentrations (MICs) of various antibiotic classes between wild-type and UbiB mutant strains, with particular attention to:

  • Aminoglycosides (membrane potential-dependent uptake)

  • Polymyxins (membrane integrity-dependent activity)

  • Quinolones (respiratory chain interactions)

Table 2: Impact of UbiB Deletion on Antibiotic Susceptibility in E. fergusonii

Virulence Connections:

• Oxidative Stress Response: Measure the ability of wild-type and UbiB mutants to:

  • Survive hydrogen peroxide challenge (similar to experiments performed with small RNAs in E. fergusonii )

  • Detoxify reactive oxygen species

  • Persist in phagocytes

• In Vivo Competition Assays: Perform mixed infections with wild-type and UbiB mutant strains to assess competitive fitness in animal models.

• Metabolic Adaptation: Characterize metabolic profiles of wild-type and UbiB mutants using:

  • Metabolomics under aerobic and anaerobic conditions

  • Respiratory chain activity measurements

  • Carbon source utilization patterns

This integrated approach will allow researchers to determine whether UbiB's role in ubiquinone biosynthesis directly impacts antibiotic resistance mechanisms and virulence traits in E. fergusonii, potentially identifying novel therapeutic targets.

What are the promising areas for future research on E. fergusonii UbiB and ubiquinone biosynthesis pathways?

Several promising research directions emerge from current understanding of E. fergusonii UbiB and ubiquinone biosynthesis:

Structural Biology Frontiers:

• High-Resolution Structures: Determine crystal or cryo-EM structures of E. fergusonii UbiB alone and in complex with substrates, ATP, and partner proteins to elucidate the catalytic mechanism and protein-protein interaction interfaces.

• Conformational Dynamics: Investigate conformational changes associated with ATP binding and hydrolysis using hydrogen-deuterium exchange mass spectrometry (HDX-MS) or single-molecule FRET techniques.

• Membrane Integration: Characterize the membrane topology and lipid interactions of UbiB using advanced techniques such as nanodiscs combined with crosslinking mass spectrometry.

Functional Integration:

• Pathway Crosstalk: Investigate potential regulatory connections between O₂-dependent and O₂-independent ubiquinone biosynthesis pathways, particularly how cells coordinate pathway selection in response to fluctuating oxygen levels .

• Broader Metabolic Context: Explore links between ubiquinone biosynthesis and other cellular processes such as phospholipid metabolism , oxidative stress response, and central carbon metabolism.

• Evolutionary Adaptations: Compare ubiquinone biosynthesis mechanisms across diverse proteobacteria to understand evolutionary adaptations to different ecological niches and oxygen availability.

Translational Applications:

• Targeted Antimicrobials: Develop inhibitors specifically targeting the O₂-independent ubiquinone biosynthesis pathway as potential antibiotics for treating infections in anaerobic tissue environments.

• Biotechnological Applications: Engineer E. fergusonii strains with enhanced ubiquinone production for biotechnological applications, leveraging the ability to produce ubiquinone under both aerobic and anaerobic conditions.

• Diagnostic Markers: Explore the potential of UbiB as a diagnostic marker for distinguishing E. fergusonii from other Escherichia species in clinical samples, possibly in combination with species-specific aspects of other pathway components.

Methodological Innovations:

• In Situ Activity Probes: Develop chemical probes that can report on UbiB activity within living bacterial cells under various conditions.

• Reconstituted Minimal Systems: Create fully defined, reconstituted ubiquinone biosynthesis systems to dissect the precise role of each component, including UbiB.

• Single-Cell Analysis: Apply single-cell techniques to understand cell-to-cell variability in ubiquinone biosynthesis and its relationship to antibiotic tolerance and persistence.

These research directions collectively promise to advance our understanding of fundamental aspects of bacterial metabolism while potentially opening new avenues for antimicrobial development and biotechnological applications.